Composition of multipurpose high functional alkaline solution composition, preparation thereof, and for the use of nonspecific immunostimulator

ABSTRACT

Disclosed are a multipurpose, high-functional, alkaline solution composition, preparation therefor and use thereof as a nonspecific immunostimulator. The composition comprises 1-25 parts by weight of borax (Na 2 B 4 O 7 .10H 2 O), 10 −5 -10 −4  parts by weight of sodium thiosulfate (Na 2 S 2 O 3 .5H 2 O), 30-150 parts by weight of potassium carbonate, 30-200 parts by weight of refined sugar (C 12 H 22 O 11 ), and 100-200 parts by weight of water, based on 100 parts by weight of sodium metasilicate (Na 2 SiO 3 .5H 2 O). In addition to bringing about an improvement in disease resistance, weight gain rate, crop yield, crop quality, harvest time, the composition shows nonspecific immunostimulating activities, including antibody production and immune enhancement, by activating immune cells, thereby maximizing vaccination effects on malignant viral diseases.

This application is a divisional application of U.S. application Ser.No. 09/755,020, filed Jan. 8, 2001 now U.S. Pat. No. 6,447,810, whichclaims priority from Korean Application No. 2000-70054, filed Nov. 23,2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a multi-purpose, high-functional,alkaline solution composition, a preparation method therefor, and usethereof as an nonspecific immunostimulator. More particularly, thepresent invention relates to an alkaline solution composition whichmostly comprises sodium metasilicate (pentahydrate) and showsnonspecific immunostimulating activities, including antibody productionand immune enhancement, by activating immune cells, thereby maximizingvaccination effects versus malignant virus diseases, preparationtherefor, and use thereof as an immunostimulator.

2. Description of the Prior Art

The functionality of alkaline mass in the body has been of greatinterest since the early 20^(th) century. Extensive research hasrecently revealed that alkaline mass in the body increases ionizationratios of potassium and sodium to heighten the purification capabilityof blood, resulting in blood clearance, fatigue-reduction, and agingretardation.

One alkaline solution composition is disclosed in Korean Pat. No.128,110, yielded to the present inventor, which comprises 10-18 parts byweight of sodium silicate, 0.1-0.5 parts by weight of sodium hyperoxide,5-10 parts by weight of potassium carbonate, 1 part by weight of sodiumcarbonate, 10-18 parts by weight of refined sugar and 0.1-3.0 parts byweight of silver thiosulfate in water. The said composition is now usedfor the post-treatment of fiber products and the fermentation offeedstuff and in the agricultural industry by virtue of its high farinfrared radiation efficiency, antibacterial activity and deodorizingactivity. The composition, however, is disadvantageous in that itspreparation is complicated and it is difficult to store for a longperiod of time.

Meanwhile, the amount of antibiotics used each year in the world isincreasing. However, the more antibiotics are used, the greater the sideeffects are. For example, higher dosages of antibiotics are needed totreat patients who have overused prescription antibiotics because theyhave become resistant to antibiotics. Furthermore, misuse and abuse ofantibiotics has resulted in the appearance of super-bacteria which arecompletely immune to existing antibiotics. In an effort to curtail theuse of antibiotics, consideration has been taken of the generalenhancement of the immune system in the body, which leads to animprovement in vaccination effects. For example, nonspecificimmunostimulators (hereinafter, referred to as “NIS”), which induce thebody to increase its immune response to external pathogens, are now ofgreat interest to medical personnel and extensive and intensive researchhas been focused on the development of NIS over the world.

In Japan, an ingredient extracted from edible mushroom (Lentinusedoddes) was reported to show an anti-cancer effect. In addition, NISwas observed in bacteria one hundred years ago. Along with the effectsof forming antibodies and inducing cytokines, the immune enhancement ofNIS has recently been under active study. For instance, a cell wallextract from Norcardia opaca is reported to induce the activation of themacrophages derived from murine peritoneal cavities (Barot-Ciobaru etal., 1987). RU41740, derived from Klebsiella pneumoniae, KP-40, derivedfrom Propionibacterium avidum, and QH-B, derived from Quillajasaponaria, are found to have useful functions in association with theinduction of cytokines and the stimulation of immune cells (Bessler etal, 1997); Nimier et al., 1999; Ronnberg et al., 1997; Siwiki et al.,19988; Tewari et al., 1996). Recently, bacterial DNA-derived CPG motifs,called immunostimulatory motifs, have been revealed to effectivelyinduce the expression of IL-6, IL-12, IL-18 and IFN-γ in immune cells(Bohle et al., 1999; Klinman et al., 1999; Krieg, 1999).

These and other NIS developed thus far suffer from the problems of beingproduced in complicated processes, inconvenient for long-term storage,and expensive.

DISCLOSURE OF THE INVENTION

With problems encountered in prior arts in mind, the present inventionhas an objective of providing an alkaline solution composition, which iseasy to produce and store for a long period of time, has inhibitoryactivity against bacteria and virus proliferation, and can serve as anonspecific immunostimulator.

It is another object of the present invention to provide a method forpreparing such an alkaline solution composition.

It is a further object of the present invention to provide uses of thealkaline solution composition.

It is still a further object of the present invention to provide methodsfor improving the weight gain rate of livestock and the yield of crops.

It is still another object of the present invention to provide a methodfor keeping agricultural products, fishes or meats fresh for a longperiod of time.

It is still yet another object of the present invention to provide anontoxic, nonspecific immunostimulator with an anticancer effect.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 is a graph in which changes in the proportions of porcine CD4⁺ Tlymphocytes in the BARODON®-fed groups and in the control group areplotted against time;

FIG. 2 is a graph in which changes in the proportions of porcine MHCclass II-presenting cells in the BARODON®-fed groups and in the controlgroup are plotted against time;

FIG. 3 is a graph in which changes in the proportions of porcine NonT/Non B lymphocytes (N lymphocytes) are plotted against time;

FIG. 4 is a graph in which changes in the proportions of porcine CD8⁺ Tlymphocytes in the BARODON®-fed groups and in the control group areplotted against time;

FIG. 5 is a histogram showing the lymphoproliferation activities ofporcine lymphocytes isolated from the peripheral blood of theBARODON®-fed groups and the control group in response to stimulationwith Con A, PHA, PWM and LPS;

FIG. 5 is a histogram showing the lymphoproliferation activities ofporcine lymphocytes isolated from the mesenteric lymph nodes of theBARODON®-fed groups and the control group in response to stimulationwith Con A, PHA, PWM and LPS;

FIG. 7 is a graph showing anti-cholera antibody productivities of theBARODON®-fed groups and the control group;

FIG. 8 is a graph in which changes in muscle Ca-ATPase activity of codsin the BARODON®-treated groups and the control group are plotted againststorage time;

FIG. 9 is an NMR spectrum showing an absorption wavelength band for ¹⁷Oof tap water; and

FIGS. 10 and 11 are NMR spectra showing absorption wavelength bands for¹⁷O of water molecules in an aqueous solution containing the compositionof the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention contemplates an NIS based on an alkaline solutioncomposition. This composition comprises sodium metasilicate, borax,sodium thiosulfate, potassium carbonate, refined sugar, and water,optionally sodium chloride, silver thiosulfate and/or sodium molybdate.

Sodium metasilicate, useful in the present invention, has fivecrystalline water molecules and comprises silicon dioxide (SiO₂) at anamount of 27.5-29.0% and sodium oxide (Na₂O) at an amount of 28.5-30.0%with a content error margin less than 2%. This compound is quite stablecompared with commercially available liquid sodium silicate. Existing asa white powder or grain, sodium metasilicate is easy to weigh accuratelyand convenient for long-term storage and transportation. When beingdissolved in water, sodium metasilicate is of strong alkalinity.Silicon, like the other constituents of sodium metasilicate, is anessential element for the growth of animals and plants.

Borax having with ten crystal water molecules has a specific density of1.715. As a constituent of borax, boron (B) is a trace element in whichanimals and plants are likely to be deficient. Fruit trees, when soilsare insufficient in boron, do not bear well and, if produced, many oftheir fruits are defective. Generally, borax is used as a bacteriumrepellent, an insect repellent, a styptic, and a glaze material. Whenmelted together with metals, borax has the property of dissolving themetals. A solution of borax dissolved in water is alkaline withapproximately pH 9.5. In the composition of the present invention, theamount of borax is preferably adjusted within the range of 1-15 parts byweight based on 100 parts by weight of sodium metasilicate. Forinstance, if borax is used at an amount less than 1 part by weight, noeffects thereof are exhibited. On the other hand, if the borax contentexceeds 15 parts by weight, toxicity may be generated.

With five crystal water molecules, sodium thiosulfate is not dissolvedin alcohol, but dissolved in water, giving a characteristic salty taste.Its aqueous solution is neutral with a pH in the range of 6.5-8.0. Witha chemical property of dissolving silver halide or other silver salts,sodium thiosulfate is generally used to extract silver from ores.Additionally, sodium thiosulfate finds applications in the removal ofchlorine and heavy metals. In the present invention, sodium thiosulfateis used at an amount of approximately 10⁻⁵-10⁻⁴ parts by weight based on100 parts by weight of sodium metasilicate. For instance, no additionaleffects can be obtained when the amount of sodium thiosulfate is lessthan the lower limit. On the other hand, an amount exceeding the upperlimit of sodium thiosulfate, when being applied to animals, acts tosediment tissular Ca, causing the animals to be excited and to haveloose bowels.

Potassium carbonate is dissolved well in water and its solution is ofstrong alkalinity with pH 11.6. Like sodium, potassium is an essentialelement for the body, playing an important role in metabolism and bloodcirculation. For example, an appropriate balance between potassium andsodium in vivo is needed to prevent hypertension and diabetes mellitus.In the present invention, a preferable amount of potassium carbonatefalls within the range of 30-150 parts by weight based on 100 parts byweight of sodium metasilicate. When being applied to animals or plants,for example, an amount outside this range of potassium carbonate mayupset the in vivo balance of sodium/potassium.

In the composition of the present invention, refined sugar preventsionized inorganic materials from recombining so as to stabilize thecomposition. In addition, refined sugar converts inorganic materials toorgano-type materials in sugar-associated forms, as well as acting toimprove the adhesiveness or adsorption of the composition. Within theamount range of 30-200 parts by weight based on 100 parts by weight ofsodium metasilicate, refined sugar can perform these roles.

Sodium chloride, an optional compound in the composition of the presentinvention, is used as a source of sodium to control the ratio ofsodium/potassium. As for silver thiosulfate, it is often used tosuppress the ethylene synthesis of plants or to facilitate thedifferentiation of plants. In an aqueous solution, silver thiosulfateforms [Ag(S₂O₃ ²⁻)₂]³⁻ at a low concentration of S₂O₃ ²⁻ while forming[Ag₂ (S₂O₃ ²⁻)₆]¹⁰⁻ at a high concentration of S₂O₃ ²⁻. In the presentinvention, silver thiosulfate, existing in a polyvalent anionic formlike sodium thiosulfate, acts to facilitate cell differentiation. In thepresent invention, sodium molybdate serves as a source of molybdenum,which is an element which animals and plants are likely to lack. Theoptional compounds are each used at an amount of 10⁻¹ parts by weight orless based on 100 parts by weight of sodium metasilicate.

After complete dissolution of the constituents, the composition of thepresent invention is tinged with ivory. The composition is odorless andnontoxic in addition to being very stable with a specific viscosity of1.43-1.50, maintaining pH 13 and ranging in viscosity from 61.0-239.0.Particularly, even when being treated with HCl, the composition of thepresent invention hardly undergoes solidification or pH changes.

Various experimental results obtained over a long period of timedemonstrate that the composition of the present invention increasesweight-gaining rates in livestock and the yield from crops, as well asserving as an excellent NIS in both plants and animals.

The composition of the present invention may be fed to livestock, alongwith feedstuff, for example, in a mixture with feedstuff or after beingfermented in feedstuff, or as a dilution with water. After feeding, thecomposition brings about excellent immune enhancement in the livestock.For example, the composition of the present invention is effectivelypreventive of porcine epidemic diarrhea (PED) and hog cholera, whichcause serious problems for the hog raising industry. Also, thecomposition was found to be effective for the prophylaxis and treatmentof fowl typhoid, which shows a high mortality, causing an enormous lossin the poultry industry. In the case of milk cows fed with thecomposition, the somatic cell count per volume of milk, which is anindex determining milk quality, was decreased. Other effects of thecomposition of the present invention on livestock include growthfacilitation and improvement in flesh quality. Sheds in which thelivestock lived while being fed with the composition of the presentinvention were found to have less offensive odors than those in whichthe livestock lived while being fed with ordinary feedstuff.

For plants, the composition of the present invention, which is appliedin mixtures of fertilizers or as dilutions in water, exhibits usefuleffects, including facilitation of germination and growth, improvementin disease resistance, increased crop yields, improvement in cropquality, etc.

A better understanding of the present invention may be obtained in lightof the following examples which are set forth to illustrate, but are notto be construed to limit the present invention.

PREPARATION EXAMPLE 1

In purified water (500 kg) maintained at 60-80° C., sodium metasilicate(pentahydrate, 300 kg), borax (decarbohydrate, 35 kg), sodiumthiosulfate (0.01 kg), sodium chloride (1 kg), and potassium carbonate(150 kg) were sequentially added, and stirred for 3 hours fordissolution. To the homogeneous solution was added refined sugar (450kg), followed by stirring for 4 hours to give an alkaline solution withpH 13 (hereinafter referred to as “BARODON®-1”).

PREPARATION EXAMPLE 2

BARODON®-1 (1,436 kg), prepared in Preparation Example 1, was dropwiseadded with a solution of silver thiosulfate (0.02 kg) in water (1 L),stirred and maintained at about 50° C. for 4 hours in an incubator. Theresulting solution was designated “BARODON®-2”.

PREPARATION EXAMPLE 3

In purified water (5 L) maintained at 100° C., sodium molybdate(Na₂MoO₄A2H₂O, 0.3 kg) was added with stirring to give a colorless,odorless solution, which was then dropwise added to BARODON®-1, stirred,and maintained at 50° C. for 4 hours in an incubator. The resultingsolution was designated “BARODON®-3”.

PREPARATION EXAMPLE 4

Using 6 kg of borax and 300 kg of potassium carbonate, an alkalinesolution was obtained in a manner similar to that of PreparationExample 1. To the alkaline solution (1,557 kg), a solution of silverthiosulfate (0.02 kg) in water (1 L) was dropwise added, followed bystirring and standing at 50° C. for 4 hours in an incubator. Theresulting alkaline solution was designated “BARODON®-4”.

PREPARATION EXAMPLE 5

Using 150 kg of refined sugar, the procedure of Preparation Example 4was repeated to give an alkaline solution with a relatively lowviscosity (hereinafter referred to as “BARODON®-5”).

EXAMPLE 1 Effect on Rice Cultivation

BARODON®-1 to -5 were tested for immune enhancement and growthfacilitation effects on the cultivation of rice plants on paddy fields.

BARODON®-1 to -5 were each diluted in 10 volumes of water and 500-folddiluted again with water. Just after being immersed for 24 hours in thediluted solution, rice seeds were sown on seedbeds. A couple of daysbefore the transplantation of rice plants from the seedbeds to paddyfields, the diluted solutions were sprayed over the nursery rice plantsin an ordinary manner. The spraying of the diluted solution wasconducted again a couple of weeks before rice booting (rice earing).

On seedbeds, the nursery rice plants treated with the composition of thepresent invention grew more uniformly and strongly and suffered almostno damages due to cold weather, compared with a control group which wasnot treated with the composition. Also, the nursery rice plants treatedwith the composition were observed to completely root only three daysafter the transplantation. No diseases, including damping-off, leafblast, and sheath blight, effected the treated rice plants during theirgrowth on field. In addition, the treated rice plants were improved inlodging-resistance and were found to suffer no lodging following atyphoon, while 25% of the control was lodged.

Amounts of produced rice were measured and the results are given inTable 1, below.

TABLE 1 Composition Crop yield per km² (Chuchung ® Rice) Control 575 kgBARODON ®-1 655 kg BARODON ®-2 690 kg BARODON ®-3 710 kg BARODON ®-4 680kg BARODON ®-5 670 kg

EXAMPLE 2 Effect on Pear Tree Cultivation

BARODON®-3 was mixed with 9 volumes of water and then re-diluted 500times. After applying dung-manure and organic fertilizers on a pearorchard, the dilution was sprayed thereon. Thereafter, a roundabout wasinstalled along the circumference of the orchard to prevent theinterference of animals. About a couple of weeks before the blooming ofpear blossoms, BARODON®-2 was sprayed on the land. The pear treesfertilized with the compositions of the present invention burst intobloom about 4-5 days earlier and produced harvestable fruits about 15days earlier than did a control, which was not fertilized with thecompositions. The pears harvested from the treated trees were graded thehighest in appearance, size and Brix degree. The pears harvested fromthe trees fertilized with the compositions were 60% reduced in blackspot occurrence and smoother in appearance, compared with the control.No soggy pears were found in the treated trees. The pear trees treatedwith the compositions of the present invention showed a fruit drop ratedue to typhoon 20% less than that of the control. The pears of thetreated trees were about 15% greater in size compared with those of thecontrol. The Brix degree of the pears of the treated trees was measuredto range from 13.0 to 15.0, which was about 12% greater than that of thecontrol. The storage period was also improved.

EXAMPLE 3 Effect on Plant Growth

A 16-fold dilution of BARODON®-3 in water was further diluted by thedilution factors given in Table 2, below and then sprayed over leaves ofspecimen plants. The leaves were found to be greater by 15.6% in length,8.7% in width, and 7-47% in foliage fresh weight, compared with acontrol group which was not treated with the solution.

TABLE 2 Dilution Factor Non- Pot No. 200-Fold 300-Fold 400-Fold SprayingLeave Width at Maximum (cm) 9^(th) day after 1 6.1 6.1 6.2 5.4 Spraying2 6.0 6.3 6.2 5.9 (10, July) Avg. 6.1 6.2 6.2 5.7 Foliage Fresh Weight(g/pot) 9^(th) Day After 1 57.8 59.2 44.6 39.9 2^(nd) Spraying 2 56.360.5 42.8 41.5 (10, July) Avg. 57.1 59.9 43.7 40.7 (140%) (147%) (107%)(100%)

PREPARATION EXAMPLE 6

BARODON®-2, prepared in Preparation Example 2, was diluted by a factorof 10 and 500 g of the resulting dilution was sprayed over 1 ton ofcombined feedstuff to give a high-functional feedstuff (hereinafterreferred to as “BARODON®-6”).

PREPARATION EXAMPLE 7

A 10-fold dilution of BARODON®-2, prepared in Preparation Example 2, inwater (10 L) was added, together with refined sugar (3 Kg), sodiumchloride (1 Kg) and water (75 L), to combined feedstuff (1 ton), afterwhich the resulting formulation was fermented for 24 hours with stirringto give high-functional feedstuff (hereinafter referred to as“BARODON®-7”).

EXAMPLE 4

The BARODON® series was tested for weight gain and immune enhancementeffects on animals as follows.

(1) Weight-Gaining Effect on Hog

30 heads of three-way cross hybrid (Yorkshire×Landrace×Durroc),fattening hogs, each of which was 15 weeks (104±4 days) old, wereselected. They were all admitted to 3 hog pens, each with a size of 4M×4.2 M, at a population of 10 heads per hog pen and adapted to the newcircumstance for one week before testing.

BARODON®-6, prepared in Preparation Example 6, was fed to a group of 10hogs (designated “Tx-1 group”) in one hog pen for 9 weeks while thefeedstuff containing 3% of BARODON®-7, prepared in Preparation Example7, was given to a group of 10 hogs (designated “Tx-2 group”) in anotherhog pen. For a control group, the same feedstuff, but lacking “BARODON®”was supplied. Thereafter, all the hogs were fed with common feedstuff.During the testing period, the hogs were all allowed free access tofeedstuff.

A measurement was made of weight gain and feedstuff intake for 6 weeksfor each group and, from the measured values, the feed efficiency(feedstuff intake/weight gain) was calculated. The average daily weightgain was measured to be 842.86 g for the control group, 890.48 g for theTx-1 group and 880.95 g for the Tx-2 group, the latter two groups being5.65% and 4.52% improved, respectively, compared with the control group.As for average daily feedstuff intake, 2.71 kg of feedstuff was consumedby the control group, 2.77 kg of feedstuff by the Tx-1 group and 2.65 kgof feedstuff by the Tx-2 group. Therefore, the control group showed afeed conversion rate of 3.22 while the Tx-1 group was calculated to be3.11 in feed conversion rate with an improvement of 3.54% and the Tx-2group grew at a feed conversion rate of 3.01, which was 6.98% improved.

(2) Perception of Pork

The pork of each of the test groups was tasted by healthy men and womenand they were interviewed regarding taste and flesh quality.

TABLE 3 Softness of Flesh Taste Soft Tough Good Moderate Control 0 8 0 3Tx-1 6 1 8 2 Tx-2 11  0 10  0 * numbers of the persons who responded tothe questions after a sampling party.

(3) Distribution of Immune Cells in Peripheral Blood of Hog

Using pig leukocyte surface-specific monoclonal antibodies and a flowcytometry such as that manufactured by Dickinson Immunocytometry System,San Jose, Calif., U.S.A., identified as FACSCalibur, proportions ofmajor histocompatibility complex (MHC) expressing cells and lymphocytesubpopulations were examined in the peripheral bloods of the Tx-1 andthe Tx-2 group as well as the control group.

(3-1) Isolation of Leukocyte from Peripheral Blood

The blood taken from the swine fore vena cava was well mixed with acidcitrate dextrose (ACD)-ethylenediamine tetraacetic acid (EDTA) and laidover a layer of Hypaque Ficoll (Histopaque, Sigma, St. Louis, Mo.,U.S.A.). After centrifugation at 1,500 rpm for 30 min, leukocytes weretaken, washed three times with phosphate buffered saline (PBS, pH 7.2),and suspended in an RPMI-1640 medium (GibcoBRL, Grand Island, N.Y.,U.S.A.). For examination, leukocyte cell counts were adjusted to 1×10⁷cells/ml while viable cells were counted according to the tryphan blueexclusion technique.

(3-2) Monoclonal Antibodies for Examination of Leukocyte Subpopulation

Effects on porcine immunological properties, including immune cellpopulations, were determined using monoclonal antibodies specificallyreactive with cell surface molecules of porcine leukocytes, i.e., MHCclass I antigens, MHC class II antigens, Po(porcine)CD2, PoCD4, PoCD8,surface IgM (sIgM), Non T/Non B (γδ TCR), and granulocyte and monocyte(G+M), as shown in Table 4, below.

TABLE 4 Isotype MAb* of MAb Molecules** Cell type*** Reference PT85AIgG_(2a) MHC class All nucleated Davis et al. (1987) I cells H42AIgG_(2a) MHC class Ag-presenting Davis et al. (1987) II cells TH81A5IgG_(2a) MHC class Ag-presenting Davis et al. (1987) II cells MSA4IgG_(2a) PoCD2 T cells Davis et al. (1987) PT90A IgG_(2a) PoCD4 T h/icells Davis et al. (1987) PT81B IgG_(2a) PoCD8 T c/s cells Davis et al.(1987) Plg45A IgG_(2b) slgM B cells Davis et al. (1987) PT79A IgG_(2a)γδTCR N cells Davis et al. (1987) Gran- Gran- Davis et al. (1987) DH59BIgG₁ ulocyte + ulocyte + Monocyte Monocyte *mab: Monoclonal antibodiesspecifically reactive with leukocyte differentiation. **Molecules:Porcine leukocyte differentiation molecules. ***Cell type: Cellsexpressing molecules.

(3-3) Flow Cytometry

Using a flow cytometry CellQuest program, proportions of leukocytesubpopulations were analyzed according to the method of Davis et al(1990). In order to take advantage of a flow cytometry using a laserbeam, cells were labeled with one or two fluorescent dyes such asfluorescein isothiocyanate (FITC) and phycoerythrin (PE) in an indirectmanner. In each well of a V-bottom 96-well microplate, 100 μl of theleukocytes isolated from blood (cell density 1×10⁷ cells/ml) was added,along with 50 μl of a monoclonal antibody (concentration 15 μg/ml) andsensitized for 30 min at 4° C., followed by washing three times with afirst washing buffer [PBS 450 ml, ACD 50 ml, 20% NaN₃ 5 ml, gammaglobulin free horse serum (GibcoBRL) 10 ml, 250 mM EDTA 20 ml, 0.5%phenol red 1 ml] through centrifugation. After decanting thesupernatant, the cell pellet on the bottom was suspended using a platemixer or a vortex mixer (Scientific Industries, Bohemia, N.Y., U.S.A.).

In a single dyeing test, an FITC-conjugated goat anti-mouse IgG+IgMantibody (Caltag Lab, U.S.A.), serving as a secondary antibody, wasdiluted by a factor of 200 and added at an amount of 100 μl to each wellin which the suspended leukocytes were contained. After sensitizationfor 30 min at 4° C., washing was conducted three times with a secondwashing buffer, which was the same as the first washing buffer, butbeing free of the horse serum, by centrifugation. A 2% PBS-formalin (38%formalin 20 ml, PBS 980 ml) solution was added at an amount of 200 μl toeach well to fix the cells.

In a double dyeing test, PoCD4 (FITC) was coupled with PoCD8 (PE), PoCD4(FITC) with MHC class II (PE), and PoCD8(FITC) with MHC class II (PE) todye cells. In detail, leukocyte cells in one well were mixed with a pairof monoclonal antibodies and primarily sensitized according to theresult of the single dyeing test, followed by washing three times withthe first washing buffer at 4° C. Thereafter, a goat antibody specificfor each monoclonal antibody isotype was used at a concentration of 1.0μg per well for FITC conjugates and at a concentration of 0.1 μg perwell for the PE conjugate, followed by secondary sensitization for 30min at 4° C. Washing and fixing procedures were conducted in the samemanner as in the single dyeing test.

After completion of the dyeing, cells were stored at 4° C. in a dark,cold space until examination. Using a flow cytometry, 2,000 or more dyedcells were analyzed to count positive-response cells. In regard to themeasurement and data analysis, FACScalibur and CellQuest program (BectonDickinson) was employed.

The results showed that the proportion of CD4⁺ T lymphocytes in porcineperipheral blood started to increase after three weeks from the feedingwith BARODON®-containing feedstuff, as shown in FIG. 1 and, on the8^(th) week after the feeding, considerably higher levels of the CD4⁺ Tlymphocytes were maintained by the Tx-1 and the Tx-2 groups than by thecontrol group (p<0.05). Particularly, the Tx-1 group maintained CD4⁺ Tlymphocytes at high levels over a period from the 8^(th) week to the13^(th) week post-application (p<0.05).

As for CD8⁺ T lymphocytes, a high level was detected in the Tx-2 groupon the third week after the feeding (p<0.01), but no noticeabledifferences were found from the 8^(th) week after the feeding, comparedwith the control group (p<0.05), as shown in FIG. 4.

Cells expressing MHC class II antigens, mainly macrophages, were countedat a considerable high level for the Tx-1 group at the 11^(th) weekafter the feeding (p<0.05) and for the Tx-2 group at the 8^(th) weekafter the feeding, compared with the control group, as shown in FIG. 2.

The count of Non T/Non B lymphocytes (N lymphocytes) in the Tx-2 groupwas maintained higher than that in the control group (p<0.01) from the3^(rd) week after the feeding, as shown in FIG. 3, showing thepossibility of enhancing both nonspecific and specific immune defenseresponses. Over a period from the 11^(th) week to the 13^(th) week afterthe feeding, the Non T/Non B lymphocytes were maintained at considerablyhigher levels in the Tx-2 group than in the control group (p<0.01).

Comparing the two BARODON®-fed groups, the Tx-2 group stayeddistinctively predominant over the Tx-1 group in MHC-class IIantigen-expressing cell level during a period from the 3^(rd) week tothe 8^(th) week after the feeding (p<0.05) as shown in FIG. 2. A littlemore CD4 and CD8 expressing cells were counted in the Tx-2 group than inthe Tx-1 group on the 3^(rd) week after the feeding (p<0.1), as shown inFIGS. 1 and 4. Over the Tx-1 group, the Tx-2 group attained noticeablesuperiority in Non T/Non B lymphocytes on the 13^(th) week after thefeeding, as shown in FIG. 3.

(4) Effect on Activity of Blood and Lymph Nodal Lymphocytes

In order to examine activities of blood and lymph nodal lymphocytes,their proliferative responses were determined by measuring[³H]-thymidine incorporation of porcine lymphocytes obtained fromperipheral blood and mesenteric lymph nodes after stimulation withConcanavalin A (Con A), Phytohemagglutinin (PHA), Pokeweed mitogen (PWM)and Lipopolysaccharide (LPS).

When being stimulated with PWM, the lymphocytes obtained from theperipheral blood of the Tx-2 group after 8 weeks of the feeding with theBARODON®-containing feedstuff were observed to be highly proliferativeas proven by a significantly larger stimulation index for the Tx-groupthan for the control group (p<0.05). At 11^(th) week post-feeding, thelymphocytes isolated from the peripheral blood of both the Tx-1 groupand the Tx-2 group gave greater proliferative responses to thestimulation with each of PHA, PWM and LPS than those isolated form thecontrol group, showing higher SI values (p<0.01), as depicted in FIG. 5.

As for lymphocytes isolated from mesenteric lymph nodes, significantlyhigher SI values were observed in both the Tx-1 group and the Tx-2 groupin response to the stimulation with PHA (p<0.05) and PWM (p<0.01) at the₈th week after the feeding than in the control group. At 11 weekspost-application, the Tx-1 group showed higher Con A- and PHA-stimulatedlymphoproliferative responses than the control group (p<0.01) while theTx-2 group was measured to have significantly high SI values in responseto the stimulation with Con A, PHA and PWM (p<0.05), as depicted in FIG.6.

(5) Effect on Proportions of Splenic and Lymph Nodal CD4⁺ CD8⁺ TLymphocyte Subpopulations

Immunohistochemistry was used to analyze proportions of T lymphocytes.After being immunologically dyed by use of the ABC method, CD4+, CD8+,and CD4+CD8+dpp T lymphocyte cell counts in mesenteric lymph nodes andspleens were measured with the aid of an image analyzer (Olympus,U.S.A.). The immunohistochemical analysis results are given in Tables 5and 6, below. In spleens, as apparent from Table 5, an increase was seenin the proportion of CD4⁺ T lymphocytes for the Tx-2 group only whileCD8⁺ and CD4⁺ CD8⁺ T lymphocyte counts were significantly increased forthe Tx-1 and the Tx-2 group, both (p<0.001). In mesenteric lymph nodes,on the other hand, both the Tx-1 and the Tx2 groups were significantlyincreased in all CD4⁺, CD8⁺ and CD4⁺ CD8⁺ dpp T lymphocyte proportions(p<0.01), as specified in Table 6, below. Particularly, such higherdistributions of T lymphocytes subpopulations were more evident in theTx-2 group (p<0.01).

TABLE 5 Group CD4⁺ CD8⁺ CD4⁺CD8⁺dpp Control 11 ± 1  8 ± 1 3 ± 1 Tx-1 11± 1 11 ± 1 6 ± 1 Tx-2 14 ± 1 17 ± 1 11 ± 1 

TABLE 6 Group CD4⁺ CD8⁺ CD4⁺CD8⁺dpp Control 32 ± 5 29 ± 2 10 ± 1 Tx-1 35± 4 39 ± 4 32 ± 3 Tx-2 40 ± 4 47 ± 5 35 ± 4

(6) Effect on Antibody Production After Vaccination with Hog CholeraVaccine

Anti-hog cholera virus IFA titers were determined after the vaccinationwith a hog cholera vaccine in BARODON®-fed groups and a non-BARODON® fedgroup. Significantly higher antibody titers were measured after threeweeks of the feeding of BARODON® in the BARODON®-fed groups (Tx-1 andTx-2) than in the control group and these titers were maintained tillthe 11^(th) week after the feeding (p<0.01), as depicted in FIG. 7.

(7) Effect on Typhoid-Resident Fowl Treated with No Antibiotics

10,000 heads of chickens infected with typhoid were fed for 10 weekswith the feedstuff containing 5 wt % of BARODON®-7, prepared inPreparation Example 7. Their mortalities by week were measured and theresults are given in Table 7, below. During the testing term, noantibiotics were administered to the typhoid-infected chickens. Asspecified in Table 7, the BARODON®-fed group showed a mortality of aslow as 0.3% on average until the 15^(th) week after the testing whereasadditional typhoid-infected 10,000 heads in a control group, which werenot fed with BARODON®, were, for the most part, dead only 4 days afterthe testing.

TABLE 7 Mortality with Lapse of Time (%) Age Week Fowl@ (week) 1 2 3 4 56 7 8 9 10 11 12 13 14 15 BARODON ®-fed 27 .34 .36 .31 .37 .30 .28 .3.27 .3 .4 .35 .33 .33 .32 .31 Control 28 1.12 1.45 1.73 7.19 almost alldead

(8) Effect on Somatic Cell Count in Milk Cow Suffering From Mastitis

In order to examine the effect of BARODON® on the somatic cell counts ofmilk cows suffering form mastitis, they were fed with the feedstuffcontaining 5 weight % of BARODON®-7, prepared in Example 7, for a coupleof months. Measurement was made of somatic cell counts. The results aregiven in Table 8, below, in which the term “liquid medicine” meansbreasts of the cows were rubbed with towels cleaned with a 100-folddilution of BARODON®-2, the term “diluted in water” means that the cowswere allowed to drink a 200-fold dilution of BARODON®-2, and thecharacter “q” stands for the number of breasts

TABLE 8 No. of Somatic Cells (× 1,000) Farm A Farm B Week Feed + LiquidDiluted Feed + Liquid After medicine Feed in Water Control medicine FeedControl Feeding (q = 10) (q = 34) (q = 6) (q = 37) (q = 16) (q = 12) (q= 36) Before 2,457 356 1,471 168   967 619 155 2 1,846 205 1,999 2761,214 886 197 (75) (57) (135) (164) (125) (150) (127) 4 1,543 144 1,683371 1,017 772 305 (62) (40) (114) (220) (105) (124) (196) 6 2,001 2282,367 517 1,137 681 353 (81) (64) (160) (307) (117) (115) (147) 8 1,788272   845 423 936 606 210 (72) (76)  (57) (251)  (96)  (97) (135)

Main pathogens causing mastitis were also counted and the results aregiven in Table 9, below.

TABLE 9 No. of Infected BARODON ®- fed Group Control Pre- Pre- PathogenFeed 4 weeks 8 weeks Feed 4 weeks 8 weeks Staphylococci sp. 11 7 6 12 1415 Streptococci sp. 2 2 1 2 3 3 Gram(−)bacilli 3 2 2 3 4 4 Fungi/Bacili0 0 0 1 2 2 Total 16 11 9 18 23 24

EXAMPLE 5

BARODON® was also investigated for its immune enhancement of the humanbody. In this regard, texts for children were manufactured with sheetsof paper, over which a 100-fold dilution of BARODON®-5, prepared inPreparation Example 5, had been sprayed, and measured for QRS waves inJapanese Far InfraRed Ray Application Institute, located in Osaka,Japan. The results are given in Table 10, below. For reference, QRS wavenumbers can give physically beneficial effects to the body when beingwithin the range of 10,000 to 20,000.

TABLE 10 Wave No. BARODON ®-S.F Paper Non-treated Paper Allergy 1,700 11Ocular Nerve 1,300 13 Autonomic Nerve 1,500 11

EXAMPLE 6 Toxicity Test

The toxicity of BARODON® was determined through in acute toxicity testsusing rats at Screening and Toxicology Center of the Korea ResearchInstitute of Chemical Technology (Test No. S-700). The acute tests wereconducted with a 10-fold dilution of BARODON®-4, according to theNational Institute of Health's Notification No. 94-3 ‘Toxicity TestingPractice for Medicines’ (Apr. 14, 1994) and the Ministry of Health andWelfare's Regulation No. 87-80 ‘Korean Good Laboratory Practice’ (Oct.29, 1987).

As a result of the test, toxic symptoms, including death, eight change,and other side effects, were not observed in males and females injectedwith the testing material while the LD₅₀ value was reported to be over5,000 mg/kg for male and female, both. This means that the compositionof the present invention is non-toxic to human bodies.

EXAMPLE 7 Mutation Test

To determine whether BARODON® acts as a mutagen, a returning mutationtest was carried out at Screening and Toxicology Center of the KoreaResearch Institute of Chemical Technology (Test No. S-694) in accordancewith the National Institute of Health's Notification No. 94-3 ‘ToxicityTesting Practice for Medicines’ (Apr. 14, 1994) and the Ministry ofHealth and Welfare's Regulation No. 87-80 ‘Korean Good LaboratoryPractice’ (Oct. 29, 1987), treating four salmonella strains (His-lessmutants of Salmonella typhimurium TA100/TA1535 (base pair substitutiontype) and TA98/TA1537 (frame shift type) with a 10-fold dilution ofBARODON®-4.

From all the four bacterial strains, negative records were observed,indicating that the test material did not cause the return mutation fromHis⁻ strains into His⁻ ones. This result means that the composition ofthe present invention is safe to the human body.

EXAMPLE 8 Effect on Cell Proliferation of Oosperm

Immature ova recovered from cow ovaries were subjected to in vitromaturation for 24 hours and then to in vitro fertilization for 20 hours.The resulting oosperms were developed for 9 days in media in which a10-fold dilution of BARODON®-4 was further diluted by factors of 500,200, 100 and 50, respectively and in control media. Bull spermiducts,obtained from a local slaughter house, and bovine oviduct epithelialcells (BOEC) and granulosa cells (GC), both taken from cow ovaries, werecultured to the third generation in the same media. Viable cells werecounted using a hemocytometer.

After the development, the externally fertilized oosperms underwentcleavage at a rate of 60.0% in the control media, at a rate of 62.4% inthe 500-fold diluted media, at a rate of 66.3% in the 200-fold dilutedmedia, and at a rate of 73.7% in the 50-fold diluted media. Developmentrates of the blastocyst embryos were measured to be 13.3% in the controlmedia, 20.0% in the 500-fold diluted media, 21.3% in the 200-folddiluted media, 18.4% in the 100-fold diluted media, and 11.0% in the50-fold diluted solution. After culturing, viable cells numbered 2.0×10⁵cells for BOEC and 3.2×10⁵ cells for GC in the control media, 2.4×10⁵cells for BOEC and 3.9×10⁵ cells for GC in the 500-fold diluted media,2.5×10⁵ cells for BOEC and 3.7×10⁵ cells for GC in the 200-fold dilutedmedia, 2.6×10⁵ cells for BOEC and 2.7×10⁵ cells for GC in the 100-folddiluted media, and 2.5×10⁵ cells for BOEC and 2.8×10⁵ cells for GC inthe 50-fold diluted media.

As demonstrated in the above data, the composition of the presentinvention has a positive influence on both the cell proliferation ofBOEC and GC and the proliferation and development of oosperms andblastocyst embryos. Particularly, blastocyst embryos were improved inproliferation rate by 50.4-60.2% when being cultured in the 500-fold and200-fold diluted media, compared with when being cultured in the controlmedia.

EXAMPLE 9 Anti-Cancer Activity

After being diluted by factors of 600-900, BARODON®-4 was tested forinhibitory activity against human tumor (cancer) cells of four kinds.

Human leukemic cells (Jurkat), human lung carcinoma cells (NCl-H69),human stroma cells (SW579), and human osteogenic sarcoma cells (U-2 OS)were obtained from the American Type Culture Collection, Rockvile, Md.,U.S.A. Each of these cells was cultured in media in which BARODON®-4 wasdiluted by factors of 600, 700, 800 and 900. Cells were examined andmeasured for their proliferation rates at each sub-culturing whileviable cells were counted using a tryphan blue method.

Prior to the examination of anticancer activity, normal cells werecultured in the same diluted media to determine whether BARODON® hascytotoxicity. The results are given in Table 11, below, demonstratingthat the diluted media did not inhibit the growth rate of humanfibroblast cells nor prevented the proliferation of hamster kidneycells.

TABLE 11 Dilution of No. of Condition of Cell lines Culture MediaBARODON ®-4 passages Cells* DMEM + 10% FCS Control  P8 ExcellentCCD-27SK 1/900  P8 Excellant (Normal skin 1/800  P8 Excellentfibroblasts 1/700  P8 Very good of human 1/600  P7 Poor DMEM + 10% FCSControl P17 Excellent BHK-21 Normal 1/900 P17 Excellent hamster 1/800P17 Very good kidney cells 1/700 P17 Good 1/600 P11 Poor *dyed with 0.4%tryphan blue and counted under a microscope

Culture results of the cancer cells of four kinds are given in Table 12,below.

TABLE 12 Dilution of No. of Condition of Cell Lines Culture MediaBARODON ®-4 passages cells* RPMI-1640 + 10% FCS Control P7 ExcellentJurkat 1/900 P7 97% Died in P7 leukemic 1/800 P7 99% Died in P7leukocytes of 1/700 P2 99% Died in P2 human 1/600 P2 100% Died in P2RPMI-1640 + 10% FCS Control P6 Excellent NCI-H69 1/900 P6 81% Died in P6Small cell 1/800 P5 98% Died in P5 carcinoma of 1/700 P2 100% Died in P2human lung 1/600 P2 100% Died in P2 RPMI-1640 + 10% FCS Control P10 Excellent SW579 1/900 P10  Excellent Thyroid 1/800 P10  Excellentcarcinoma of 1/700 P10  Very good human 1/600 P9 Good Mccoy's 5A + 15%FCS Control P10  Excellent U-20S 1/900 P10  Excellent Osteogenic 1/800P10  Very good Sarcoma of 1/700 P9 39% Died in P2** human 1/600 P9 47%Died in P2** *dyed with 0.4% tryphan blue and counted under a microscope**100% dead after P3

As apparent from Tables 11 and 12, the compositions of the presentinvention have inhibitory activity against human tumor cells whilegiving no damages to normal cells. Therefore, the present invention canbe used for the effective treatment of human tumors of some kinds eventhough the efficacy may be different depending on kinds of tumors.

EXAMPLE 10 Effect on Freshness Retention

Fresh cods were flash-frozen with dry ice before being transported to alaboratory. After being immersed in one of a BARODON®-free solution, a0.05%, 0.1% or 0.5% BARODON®-2 solution for 10 min, they were stored at0° C. for 7 days during which their freshness was examined.

As a fresh index for fishes, the activity of muscular Ca-ATPase, whichis connected with protein denaturation, was measured in the subjects.The best results were observed in the cods immersed in the 0.5%BARODON®-2 solution, indicating the retention of the original freshnessfor 5 days, as shown in FIG. 8.

EXAMPLE 11 Effect on Water Activation

Aqueous solutions in which BARODON®-5 was diluted at dilution ratios of1:2,800 and 1:5,600 with tap water were measured for ¹⁷O-NMR of H₂O. Thespectroscopic examination was conducted at 20° C. with the aid of aspectrophotometer JNM-EX270 at the Japanese Water Science ResearchMeeting, Japan, using as a control tap water. ¹⁷O absorption wavelengthbands were measured at 149.6 Hz (full width at half maximum) for thecontrol (FIG. 9), 53.6 Hz for the 2,800-fold diluted solution (FIG. 10),and 54.7 Hz for the 5,600-fold diluted solution (FIG. 11).

These spectroscopic analysis results indicated that the tap water is ofa heterologous phase, resulting from the aggregation of various watermolecule combinations which differ from one to another in the number ofbound water molecules whereas the BARODON®-containing solutions are ofnear crystalline phases in which water molecule combinations arecomposed of constant, minimal numbers of bound water molecules. Withsmaller numbers of bound water molecules, water is in a more activestate, showing better fluidity and biopermeability. Because activatedwater can be useful for the growth of animals and plants, thecomposition of the present invention can be used as a beverage additiveor a wastewater-treating agent.

EXAMPLE 12 Behavior of BARODON® Upon Neutralization

BARODON®-4, prepared in Preparation Example 4, was of strong alkalinitywith pH 13.20. When BARODON®-4 was neutralized with 0.1 N HCl, aprecipitate formed at the contact area. Further, the composition did notshow detectable pH change even after addition of a considerable amountof HCl. This behavior indicates that the composition of the presentinvention is highly reactive because of being sufficiently ionized.

While a 1,000-fold dilution of the composition was neutralized with 0.1N HCl, neither coagulates nor bubbles were observed under a microscope.In this case, a change occurred in the pH.

From this behavior of BARODON® upon neutralization, it can be recognizedthat, when being used at an appropriate amount, the composition of thepresent invention does not produce coagulates after the reaction withHCl within human and animal stomach. In addition, no gas is produced bythe introduction of the present invention in the stomach, so that thecomposition is free of the problems associated with gas production.

When being applied to animals and plants, as described hereinbefore, thecomposition of the present invention can bring about an improvement indisease resistance, weight gain rate, crop yield, crop quality, harvesttime. Also, the composition of the present invention shows nonspecificimmunostimulating activities, including antibody production and immuneenhancement, by activating immune cells, thereby maximizing vaccinationeffects versus malignant virus diseases.

With remarkable inhibitory activity against some kinds of human tumors,the composition of the present invention can be used as a therapeutic ora preventive agent.

In addition, when the composition of the present invention is fed inmixture with feedstock to livestock, the sheds in which the livestocklived were found to have less offensive odors and the feedstuff islittle infested with noxious insects. Further, the composition of thepresent invention is highly useful as a preservative for keeping foodsfresh.

The present invention has been described in an illustrative manner, andit is to be understood that the terminology used is intended to be inthe nature of description rather than of limitation. Many modificationsand variations of the present invention are possible in light of theabove teachings. Therefore, it is to be understood that within the scopeof the appended claims, the invention may be practiced otherwise than asspecifically described.

What is claimed is:
 1. A method for enhancing non-specific immunostimulating activity in the immune system of an animal or a plant, in which an effective amount of a composition comprising 100 parts by weight of sodium metasilicate (Na₂SiO₃≅H₂O), 1-15 parts by weight of borax (Na₂B₄O₇≅10H₂O), 10⁻⁵-10⁻⁴ parts by weight of sodium thiosulfate (Na₂S₂O₃≅H₂O), 30-150 parts by weight of potassium carbonate, 30-200 parts by weight of refined sugar (C₁₂H₂₂O₁₁), and 100-200 parts by weight of water or a dilution thereof is added in food, fertilizer, feedstuff, drug or water, and fed to the animal or fertilized to the plant.
 2. A method for enhancing human or animal immune defense against bacteria and viruses, in which an effective amount of a composition comprising 100 parts by weight of sodium metasilicate (Na₂SiO₃≅H₂O), 1-15 parts by weight of borax (Na₂B₄O₇≅10H₂O), 10⁻⁵-10⁻⁴ parts by weight of sodium thiosulfate (Na₂S₂O₃≅5H₂O), 30-150 parts by weight of potassium carbonate, 30-200 parts by weight of refined sugar (C₁₂H₂₂O₁₁), and 100-200 parts by weight of water or a dilution thereof is administered to humans or animals. 